Biological prostheses are medical devices that utilize animal tissues. Examples of suitable animal tissues include, for example, bovine, porcine, ovine and equine. Depending on the various medical uses, the biological tissue includes cardiac valves, pericardium, tendons, ligaments, dura mater, skin, veins, etc.
The tissues used in biological prostheses are formed primarily of collagen, a protein with a structural unit represented by three polypeptide chains that associate to form a triple helix. The collagen molecules assemble to form microfibrils that in turn assemble to form fibrils that, arranged in corrugated or parallel bundles, give rise to true collagen fibers. Such tissues have good resistance to traction and are flexible but substantially inextensible.
Animal tissues used in biological prostheses are first subjected to numerous washings to eliminate traces of blood and a careful removal of fat and ligaments. However, cells or cellular residues from the animal donor can remain trapped in the structure of the tissue itself. As a result it is possible that the immune system of the host gives rise to a rejection phenomenon that can lead to the destruction of the tissue constituting the biological prosthesis.
An additional problem is degradation of the collagenous biological tissue once implanted in the host organism. For this reason, the biological tissues are subjected to a fixation treatment that has the aim of protecting the tissue for such degradation phenomena and contributing to preventing the above-mentioned rejection phenomenon.
Among the substances used for the fixation of biological tissues, the most common is glutaraldehyde. This bifunctional molecule, carrying two aldehyde groups, is capable of stably binding together free amino groups of the amino acids that constitute the polypeptide chains both within one collagen molecule and between adjacent collagen molecules. In this way glutaraldehyde forms intra-chain and inter-chain bridge structures, causing the cross linking of the biological tissue. Such cross linking protects the tissue from degradation by the host and confers favorable mechanical properties such as for example a better resistance to traction with respect to untreated tissue.
Glutaraldehyde is a highly bactericidal and virucidal substance; therefore, in addition to cross linking the tissue, the fixation step also determines at least a partial sterilization.
In addition, glutaraldehyde is capable of binding to the free amino residues of the membrane proteins of the cellular components still present, masking their antigenic potential and impeding immune activation phenomena and rejection by the host.
In spite of widespread use, glutaraldehyde has a disadvantage of being one of the factors that favor pathological calcification of implanted tissues. The calcium, present in the bodily fluids of the host organism, accumulates on proteinaceous tissue giving rise to a process of, for example in the case of biological cardiac valves, one of the principle cause of valve failure. The calcium deposits can cause reduced flexibility of the portion of biological tissue constituting the valve (or the so-called valve leaflets or cusps) and lead to laceration of the tissue itself, causing a partial or total loss of valve function. The mechanism responsible for the calcification is not yet completely known and is attributed to numerous factors but is know that, following the process of glutaraldehyde fixation, free aldehyde groups that remain on the tissue can create binding sites for calcium.
In addition, phospholipids within the cellular membranes of the cells or cellular residues from the donor animal that remain trapped in the tissue structure also constitute sites for the binding and accumulation of calcium.
To limit the process of calcification in tissues destined for use in biological prostheses, recourse has been taken to various treatments to neutralize the aldehyde residues remaining free after the fixation step and to remove membrane phospholipids. For example, US-A-2006/0193885 describes the use of two solutions, the first containing lower alcohols to remove membrane phospholipids and the second containing arginine, histidine, lysine or salts of aspartic or glutamic acid to neutralize the aldehyde groups present on the biological tissue after fixation. U.S. Pat. No. 6,479,079 describes treatment of biological tissue for the removal of membrane phospholipids with solutions including alcohols having from 4 to 36 carbon atoms. U.S. Pat. No. 5,873,812 describes the use of amino carboxylic acids, for example homocysteic acid, to neutralize the aldehyde residues that remain free on biological tissue after the fixation process.
However, such methods are not ideal solutions because binding sites for calcium remain on the biological tissue, in particular, the neutralization of free aldehyde groups present after the process of fixation with glutaraldehyde is often partial.